Internal sensory neurons regulate stage-specific growth in Drosophila.

Animals control their developmental schedule in accordance with internal states and external environments. In Drosophila larvae, it is well established that nutrient status is sensed by different internal organs, which in turn regulate production of insulin-like peptides and thereby control growth. In contrast, the impact of the chemosensory system on larval development remains largely unclear. Here, we performed a genetic screen to identify gustatory receptor (Gr) neurons regulating growth and development, and found that Gr28a-expressing neurons are required for proper progression of larval growth. Gr28a is expressed in a subset of peripheral internal sensory neurons, which directly extend their axons to insulin-producing cells (IPCs) in the central nervous system. Silencing of Gr28a-expressing neurons blocked insulin-like peptide release from IPCs and suppressed larval growth during the mid-larval period. These results indicate that Gr28a-expressing neurons promote larval development by directly regulating growth-promoting endocrine signaling in a stage-specific manner.

Bioproduction of n-3 polyunsaturated fatty acids by nematode fatty acid desaturases and elongase in Drosophila melanogaster.

n-3 polyunsaturated fatty acids (n-3 PUFAs), including α-linolenic acid and eicosapentaenoic acid (EPA), are essential nutrients for vertebrates including humans. Vertebrates are n-3 PUFA-auxotrophic; hence, dietary intake of n-3 PUFAs is required for their normal physiology and development. Although fish meal and oil have been utilized as primary sources of n-3 PUFAs by humans and aquaculture, these traditional n-3 PUFA sources are expected to be exhausted because of the increasing consumption requirements of humans. Hence, it is necessary to establish alternative n-3 PUFA sources to reduce the gap between the supply and demand of n-3 PUFAs. Here, we investigated whether insects, which are considered as a novel source of essential nutrients, could store n-3 PUFAs by the forced expression of n-3 PUFA biosynthetic enzymes. We utilized Drosophila as an insect model to generate transgenic strains expressing Caenorhabditis elegans PUFA biosynthetic enzymes and examined their effects on the proportion of fatty acids. The ubiquitous expression of methyl-end desaturase FAT-1 prominently enhanced the proportions of α-linolenic acid, indicating that FAT-1 is useful for metabolic engineering to fortify α-linolenic acid in insect. Furthermore, the ubiquitous expression of nematode front-end desaturases (FAT-3 and FAT-4), PUFA elongase (ELO-1), and FAT-1 led to EPA bioproduction. Hence, nematode PUFA biosynthetic genes may serve as powerful genetic tools for enhancing the proportion of EPA in insects. This study represents the first step toward the establishment of n-3 PUFA-producing insects.

Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila.

Endocycle is a commonly observed cell cycle variant through which cells undergo repeated rounds of genome DNA replication without mitosis. Endocycling cells arise from mitotic cells through a switch of the cell cycle mode, called the mitotic-to-endocycle switch (MES), to initiate cell growth and terminal differentiation. However, the underlying regulatory mechanisms of MES remain unclear. Here we used the Drosophila steroidogenic organ, called the prothoracic gland (PG), to study regulatory mechanisms of MES, which is critical for the PG to upregulate biosynthesis of the steroid hormone ecdysone. We demonstrate that PG cells undergo MES through downregulation of mitotic cyclins, which is mediated by Fizzy-related (Fzr). Moreover, we performed a RNAi screen to further elucidate the regulatory mechanisms of MES, and identified the evolutionarily conserved chaperonin TCP-1 ring complex (TRiC) as a novel regulator of MES. Knockdown of TRiC subunits in the PG caused a prolonged mitotic period, probably due to impaired nuclear translocation of Fzr, which also caused loss of ecdysteroidogenic activity. These results indicate that TRiC supports proper MES and endocycle progression by regulating Fzr folding. We propose that TRiC-mediated protein quality control is a conserved mechanism supporting MES and endocycling, as well as subsequent terminal differentiation.

The presence of odd-chain fatty acids in Drosophila phospholipids.

Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.

Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster.

Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation.

Autocrine regulation of ecdysone synthesis by ß3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis.

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, ß3-octopamine receptor (Octß3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octß3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

The bioconversion of dietary α-linolenic acid to eicosapentaenoic acid in Bombyx mori.

n-3 Long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), including eicosapentaenoic acid (EPA), are essential multifunctional nutrients in animals. Microorganisms such as microalgae are known to be n-3 LC-PUFA producers in aquatic environments. Various aquatic invertebrates, including Harpacticoida copepods, and a few terrestrial invertebrates, such as the nematode Caenorhabditis elegans, possess n-3 LC-PUFA biosynthetic enzymes. However, the capacity for n-3 LC-PUFA biosynthesis and the underlying molecular mechanisms in terrestrial insects are largely unclear. In this study, we investigated the fatty acid biosynthetic pathway in the silkworm Bombyx mori and found that EPA was present in silkworms throughout their development. Stable isotope tracing revealed that dietary α-linolenic acid (ALA) was metabolized to EPA in silkworm larvae. These results indicated that silkworms synthesize EPA from ALA. Given that EPA is enriched in the central nervous system, we propose that EPA confers optimal neuronal functions, similar to docosahexaenoic acid, in the mammalian nervous system.

A Neuropeptide Signaling Network That Regulates Developmental Timing and Systemic Growth in Drosophila.

Animals sense chemical cues such as nutritious and noxious stimuli through the chemosensory system and adapt their behavior, physiology, and developmental schedule to the environment. In the Drosophila central nervous system, chemosensory interneurons that produce neuropeptides called Hugin (Hug) peptides receive signals from gustatory receptor neurons and regulate feeding behavior. Because Hug neurons project their axons to the higher brain region within the protocerebrum where dendrites of multiple neurons producing developmentally important neuropeptides are extended, it has been postulated that Hug neurons regulate development through the neuroendocrine system. In this study, we show that Hug neurons interact with a subset of protocerebrum neurons that produce prothoracicotropic hormone (PTTH) and regulate the onset of metamorphosis and systemic growth. Loss of the hug gene and silencing of Hug neurons caused a delay in larval-to-prepupal transition and an increase in final body size. Furthermore, deletion of Hug receptor-encoding genes also caused developmental delay and body size increase, and the phenotype was restored by expressing Hug receptors in PTTH-producing neurons. These results indicate that Hug neurons regulate developmental timing and body size via PTTH-producing neurons. This study provides a basis for understanding how chemosensation is converted into neuroendocrine signaling to control insect growth and development.

Expression of ß-adrenergic-like octopamine receptors during Drosophila development.

An invertebrate biogenic amine, octopamine, plays diverse roles in multiple physiological processes (e.g. neurotransmitter, neuromodulator, and circulating neurohormone). Octopamine is thought to function by binding to G-protein-coupled receptors. In Drosophila, three ß-adrenergic-like octopamine receptors (Octß1R, Octß2R, and Octß3R) have been identified. We investigated the expression of three OctßR genes in embryos, larvae, and adults. These OctßRs showed distinct expression patterns in the central nervous system (CNS) throughout development, and Octß3R expression was evident in an endocrine organ, the ring gland, in larvae. In larvae, Octß1R, Octß2R, and Octß3R were expressed in salivary glands and imaginal discs, Octß2R and Octß3R in midgut, and Octß3R in gonads. In adult, besides in the CNS, each OctßR was strongly expressed in ovary and testis. Our findings provide a basis for understanding the mechanisms by which OctßRs mediate multiple diverse octopaminergic functions during development.

Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila.

Animals develop from juveniles to sexually mature adults through the action of steroid hormones. In insect metamorphosis, a surge of the steroid hormone ecdysone prompts the transition from the larval to the adult stage. Ecdysone is synthesized by a series of biosynthetic enzymes that are specifically expressed in an endocrine organ, the prothoracic gland. At the late larval stage, the expression levels of ecdysone biosynthetic enzymes are upregulated through the action of numerous transcription factors, thus initiating metamorphosis. In contrast, the mechanism by which chromatin regulators support the expression of ecdysone biosynthetic genes is largely unknown. Here, we demonstrate that Su(var)2-10 and Su(var)205, suppressor of variegation [Su(var)] genes encoding a chromatin regulator Su(var)2-10 and nonhistone heterochromatic protein 1a, respectively, regulate the transcription of one of the heterochromatic ecdysone biosynthetic genes, neverland, in Drosophila melanogaster. Knockdown of Su(var)2-10 and Su(var)205 in the prothoracic gland caused a decrease in neverland expression, resulting in a defect in larval-to-prepupal transition. Furthermore, overexpression of neverland and administration of 7-dehydrocholesterol, a biosynthetic precursor of ecdysone produced by Neverland, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals. These results indicate that Su(var)2-10- and Su(var)205-mediated proper expression of neverland is required for the initiation of metamorphosis. Given that Su(var)2-10-positive puncta are juxtaposed with the pericentromeric heterochromatic region, we propose that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the neverland gene locus is essential for its transcriptional activation.

Influences of the Interactions of Genetic Variations of Seven Core Circadian Clock Genes with Lifestyle Factors on Metabolic Parameters.

INTRODUCTION: In mammals, circadian rhythms regulate many behavioral and physiological processes. Genetic and epidemiological studies have shown that dysregulation of the circadian rhythm induces chronic metabolic diseases, such as obesity, diabetes, and dyslipidemia. We aimed to know the interactions of genetic variations of seven core circadian clock genes with lifestyle factors on the determination of metabolic parameters. METHODS: We have analyzed the impacts of genotype of seven core circadian clock genes (i.e., CLOCK, BMAL1, PER1, PER2, PER3, CRY1, and CRY2) and lifestyle factors (i.e., physical activity and sleep duration) in 575 Japanese males on the determination of metabolic parameters (i.e., body mass index [BMI], serum glucose, glycated hemoglobin [HbA1c], low-density lipoprotein cholesterol [LDL-C], and high-density lipoprotein cholesterol [HDL-C] levels). RESULTS: We have detected the associations between genotypes of PER3 and serum HbA1c level and genotypes of CRY1 and serum LDL-C level. Additionally, the interactions of the genotypes of PER1 and PER3 with physical activity for determining BMI, the genotypes of CLOCK with physical activity for determining serum HbA1c levels were observed. Furthermore, for determining serum HDL-C levels, the interactions of the genotypes of CRY2 with physical activity or sleep duration were observed. DISCUSSION/CONCLUSION: Our findings indicate that the interactions of genotypes for core circadian clock genes and lifestyle factors (i.e., physical activity and sleep duration) are important for determining metabolic parameters.

The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila.

Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. Here, we show that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. We propose that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period.

Loss of CNDP causes a shorter lifespan and higher sensitivity to oxidative stress in Drosophila melanogaster.

Increasing oxidative stress seems to be the result of an imbalance between free radical production and antioxidant defenses. During the course of aging, oxidative stress causes tissue/cellular damage, which is implicated in numerous age-related diseases. Carnosinase (CN or CNDP) is dipeptidase, which is associated with carnosine and/or glutathione (GSH) metabolism, those are the most abundant naturally occurring endogenous dipeptide and tripeptides with antioxidant and free radical scavenger properties. In the present study, we generated Drosophila cndp (dcndp) mutant flies using the CRISPR/Cas9 system to study the roles of dcndp in vivo. We demonstrate that dcndp mutant flies exhibit shorter lifespan and increased sensitivity to paraquat or hydrogen peroxide (H2O2) induced oxidative stress. These results suggest that dcndp maintains homeostatic conditions, protecting cells and tissues against the harmful effects of oxidative stress in the course of aging.

The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in Drosophila melanogaster.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

Adult-specific insulin-producing neurons in Drosophila melanogaster.

Holometabolous insects undergo metamorphosis to reorganize their behavioral and morphological features into adult-specific ones. In the central nervous system (CNS), some larval neurons undergo programmed cell death, whereas others go through remodeling of axonal and dendritic arbors to support functions of re-established adult organs. Although there are multiple neuropeptides that have stage-specific roles in holometabolous insects, the reorganization pattern of the entire neuropeptidergic system through metamorphosis still remains largely unclear. In this study, we conducted a mapping and lineage tracing of peptidergic neurons in the larval and adult CNS by using Drosophila genetic tools. We found that Diuretic hormone 44-producing median neurosecretory cells start expressing Insulin-like peptide 2 in the pharate adult stage. This neuronal cluster projects to the corpora cardiaca and dorsal vessel in both larval and adult stages, and also innervates an adult-specific structure in the digestive tract, the crop. We propose that the adult-specific insulin-producing cells may regulate functions of the digestive system in a stage-specific manner. Our study provides a neuroanatomical basis for understanding remodeling of the neuropeptidergic system during insect development and evolution.

The Combined Effects of Genetic Variation in the CNDP1 and CNDP2 Genes and Dietary Carbohydrate and Carotene Intake on Obesity Risk.

BACKGROUND/AIMS: It is possible that carnosinase (CNDP1) and cellular nonspecific dipeptidase (CNDP2) have important roles in protecting cells and tissues against the damage of oxidative stress. Oxidative stress and subsequent inflammation are key factors in the development of common chronic metabolic diseases, such as obesity. We aimed to investigate the combined effects of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate and carotene intake on obesity risk. METHODS: A total of 1,059 Japanese men were randomly selected from participants who visited a medical center for routine medical checkups. We analyzed the relationships between the genotypes of 4 single-nucleotide polymorphisms (SNPs) (rs12605520, rs7244647, rs4891558, and rs17089368) in the CNDP1/CNDP2 locus and body mass index or prevalence of obesity/overweight taking into account dietary carbohydrate and carotene intake. RESULTS: We found that 2 SNPs (rs7244647 in CNDP1 and rs4891558 in CNDP2) were associated with obesity risk. In addition, these associations were observed only in the group with high carbohydrate and low carotene intake but not in the group with low carbohydrate and high carotene intake. CONCLUSIONS: Our findings indicate that the combination of genetic variations in CNDP1 and CNDP2 and dietary carbohydrate/carotene intake modulate obesity risk.